Co‐targeting FAK and Gli1 inhibits the tumor‐associated macrophages‐released CCL22‐mediated esophageal squamous cell carcinoma malignancy

Abstract Esophageal squamous cell carcinoma (ESCC) is a frequently seen esophageal tumor type in China. Activation of signaling proteins and relevant molecular mechanisms in ESCC are partially explored, impairing the antitumor efficiency of targeted therapy in ESCC treatment. Tumor‐associated macrophages (TAMs)‐released C‐C motif chemokine 22 (CCL22) can activate intratumoral focal adhesion kinase (FAK), thus promoting the progression of ESCC. Here, we demonstrated that highly secreted CCL22 by TAMs (CCL22‐positive TAMs) induced ESCC cell stemness and invasion through facilitating transcriptional activity of intratumoral glioma‐associated oncogene 1 (Gli1), a downstream effector for Hedgehog (HH) pathway. Mechanistically, FAK‐activated protein kinase B (AKT) mediated Gli1 phosphorylation at its Ser112/Thr115/Ser116 sites and released Gli1 from suppressor of fused homolog, the endogenous inhibitor of Gli1 to activate downstream stemness‐associated factors, such as SRY‐box transcription factor 2 (SOX2), Nanog homeobox (Nanog), or POU class 5 homeobox (OCT4). Furthermore, inhibition of FAK activity by VS‐4718, the FAK inhibitor, enhanced antitumor effect of GDC‐0449, the HH inhibitor, both in xenografted models and in vitro assays. Clinically, CCL22/Gli1 axis is used to evaluate ESCC prognosis. Overall, our study establishes the communication of FAK with HH pathway and offers the novel mechanism related to Gli1 activation independent of Smoothened as well as the rationale for the anti‐ESCC combination treatment.


INTRODUCTION
Esophageal cancer (EC) has posed a tremendous threat to human health.Esophageal squamous cell carcinoma (ESCC) occupies 90% of global EC cases, which shows a high prevalence rate in China. 1,2Extensive treatments are needed for many ESCC cases, like surgery, chemotherapy, and chemoradiotherapy.4][5][6] Some large sequencing and multi-platform studies assess the epigenetic, transcriptomic, and mutational patterns of ESCC.2][13] Our previous work has shown that tumor-associated macrophages (TAMs)released C-C motif chemokine 22 (CCL22) stimulates the activity of its receptor-C-C chemokine receptor type 4 (CCR4) in ESCC cells and subsequently activates the intratumoral focal adhesion kinase (FAK)/AKT axis to induce the malignant progression of ESCC. 14][17][18] Importantly, FAK mediates complex effects of the tumor immune microenvironment and tumor cells via activating transcriptional factors and then inducing the expression or secretion of downstream tumor-promoting molecules and TME-educating factors.Collectively, choosing FAK as the effective target is critical for the combination therapy, especially if tumor cells rely on the intercellular signalings in the milieu of TME.Multiple signaling pathways are usually manipulated by the interaction between tumor cells and their surrounding TME to control several steps during tumor malignant progression, but how they cooperate with one another to control relevant biologic behaviors and whether these signaling interactions can be targetable remain poorly understood.
0][21] Secreted HH mediates G-coupled receptor-like signal transducer Smoothened (SMO) to activate Gli, followed by nuclear translocation and stimulation of tumor-promoting gene transcription. 22Interestingly, glioma-associated oncogene 1 (Gli1) can be activated by phosphatidylinositol 3 kinase (PI3K)/AKT pathway in mesenchymal stromal cells, 23 critical for immune response in macrophages regulated by Foxo1/β-catenin axis, 24 and stimulated by mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase 1 (S6K1) signaling cascade in chondrosarcoma. 25These studies have shown that Gli1 can be activated by several intracellular signaling proteins independent of the canonical HH signaling, indicating that Gli1 may possibly be the excellent candidate for combination therapy.7][28] The more complete understanding toward Gli1-regulating mechanisms in ESCC could be beneficent for ESCC-targeted therapies.
Although both FAK and Gli1 are the critical targets for the development of antitumor agents, the correlation between intratumoral FAK and HH/Gli1 pathways in ESCC, especially in the presence of TME, has not been yet explored.In the present study, we attempted to classify the combinatorial effect of FAK and HH/Gli1 pathways inhibition on the ESCC malignancy.Correspondingly, we explored whether the CCL22/FAK signaling axis related to HH pathway activation in ESCC and the underlying mechanisms.

CCL22-positive TAMs facilitate ESCC malignancy via intratumoral CCR4
Previously, we have demonstrated that TAMs-derived CCL22 is abundantly expressed in ESCC stroma. 14We evaluated the secretion status of CCL22 in six cases (F-G) The invasive or anchorage-independent growth ability of indicated ESCC cells in the presence of rhCCL22 (50 ng/mL) was evaluated using transwell invasion assay (F) or soft agar-based colony formation assay (G).***p < 0.001 as compared with the control cells.$$$ p < 0.001 as compared with PriTAMs #1, #2, or #3.Two-tailed unpaired Student's t-test.Error bars, mean ± SD of five independent experiments. of primary TAMs using enzyme-linked immunosorbent assay (ELISA) assay and found that CCL22 is highly expressed in the conditioned media (CM) of four cases of primary TAMs (CCL22-positive TAMs), whereas the secretion of CCL22 from other two cases of primary TAMs are very low (CCL22-negative TAMs) (Figure 1A).Next, we used co-culture transwell system (pore size: 8 μm) for evaluating TAMs-mediated ESCC cell invasion through culturing KYSE410/KYSE510 cells within the top transwell chamber and TAMs within the bottom chamber (Figure 1B).CCL22-positive TAMs could effectively promote KYSE410/KYSE510 cell invasion (Figure 1C and Figure S1).Nonetheless, CCL22-negative TAMs (two cases) could not induce KYSE410/KYSE510 cell invasion (Figure 1C and Figure S1).CCL22 Ab (50 μg/mL) evidently blocked CCL22-positive TAMs-mediated ESCC cell invasion (Figure 1C and Figure S1).
Metastatic tumor cells experience the epithelialmesenchymal transition (EMT) and acquire stem cell-like phenotype. 29In our prior work, TAMs-released CCL22 effectively induced the EMT of ESCC cells. 14We further assessed whether TAMs-derived CCL22 could induce the anchorage-independent growth of ESCC cells and found that CM from CCL22-positive TAMs significantly stimulated the soft agar-based colony formation of KYSE410 and KYSE510 cells, whereas the CM from CCL22-negative TAMs not stimulated the soft agar-based colony formation of KYSE410 and KYSE510 cells (Figure 1D).Additionally, employment of CCL22 Ab (50 μg/mL) effectively suppressed CCL22-positive TAMs-induced anchorageindependent colony forming ability in indicated ESCC cells (Figure 1D).
CCR4 expressed in ESCC cells has an important effect on CCL22-induced FAK/AKT pathway activation. 14We depleted CCR4 in KYSE410/KYSE510 cells with siRNAs to observe whether CCR4 is critical for CCL22-mediated ESCC malignant progression.As the results shown in Figure 1E-G and Figure S2, depletion of intratumoral CCR4 effectively blocked recombinant human (rh) CCL22 protein (50 ng/mL)-mediated invasion and anchorageindependent growth of KYSE410 and KYSE510 cells.

CCL22 induces tumor malignancy via stimulating the intratumoral Gli1 activity
Since Gli1 is the important tumor-promoting transcriptional factor and therapeutic target, we utilized the Glidependent luciferase reporter system for evaluating effects of TAMs-released CCL22 on the intratumoral Gli1 activity and its nuclear location.CM from CCL22-positive TAMs and rhCCL22 protein (50 ng/mL) substantially increased the Gli transcriptional activity and the nuclear Gli1 expression (Figure 2A,B).To further evaluate whether CCR4 participates in CCL22-mediated Gli1 activity, rhCCL22 (50 ng/mL) was added to incubate with CCR4-depleted KYSE410/KYSE510 cells, we found that CCR4 siRNAs effectively inhibited CCL22-induced expression of nuclear Gli1 and its activity in indicated ESCC cells (Figure S3A,B).
We then evaluated Gli1 levels in 114 ESCC specimens through immunohistochemistry (IHC) assay.Seventy-six out of the 114 cases (67%) for Gli1 were positive.However, 14 out of the 66 cases (21%) for Gli1 staining in adjacent normal tissues were positive (Figure S6A).There was strongly positive correlation between the level of intratumoral Gli1 and some clinical factors, like T/N status or tumor stage (Figure S6B).As revealed by results of Kaplan-Meier curve analysis, ESCC patients harbored high Gli1 level that exhibited poor overall survival (Figure S6C).We analyzed the clinical relevance between Gli1 and stromal CCL22, and found that Gli1 level was tightly related to stromal CCL22 expression in 45 cases of ESCC tissue samples (Figure S6D).
For analyzing the role of CCL22/FAK axis in activating Gli1 function, we assessed the interaction of Gli1 with the FAK-related signalosome components. 14Without the stimulation of CCL22, no interactions were found between Gli1 and CCR4/DGKα/FAK signalosome, only slight interaction between Gli1 and AKT.However, following rhCCL22  and 2. The transfection efficacy was evaluated using immunoblotting for detection of (50 ng/mL) stimulation, Gli1 strongly interacted with AKT rather than other components in the CCR4/DGKα/FAK signalosome (Figure 3C).Because Gli1 activity is suppressed by suppressor of fused homolog (SuFu), we analyzed if CCR4/DGKα/FAK axis interacted with SuFu.As the results shown in Figure 3D, SuFu showed no interaction with any components of this signalosome, regardless of CCL22 treatment.VS-4718 (1 μM), but not cyclopamine and GDC-0449 (2.5 μM), effectively disrupted the interaction between AKT and Gli1 (Figure 3E).Correspondingly, CCR4 or FAK siRNAs significantly abolished the CCL22-induced formation of Gli1/AKT complex in indicated ESCC cells (Figure S9A,B).Taken together, the CCL22/FAK axis might regulate Gli1 via FAK/AKT axis.
Furthermore, ESCC cells harbored Gli1 S112/T115/S116E mutant had the strongest invasive and anchorageindependent growth abilities among all transfected cells (Figure S12A,B and Figure S13).Gli1 wt also facilitated the invasion and soft agar-based colony formation in KYSE410/KYSE510 cells, especially under the stimulation of CCL22-positive TAMs and rhCCL22 (Figure S12A and Figure S13).However, Gli1 S112/T115/S116A mutant decreased the invasion and anchorage-independent growth of indicated ESCC cells, compared with control vector (Figure S12A,B and Figure S13).
Importantly, IHC analysis in 114 cases of clinical ESCC samples showed that pGli1 Ser 112 /Thr 115 /Ser 116 was abundantly expressed in tumor cells (71%; 81 out of 114), but it showed negative expression within adjacent noncarcinoma esophageal samples (24%; pGli1 positive samples were 16 out of 66) (Figure S14A).Strong expression of pGli1 Ser 112 /Thr 115 /Ser 116 was positively related to several clinical parameters, including advanced stage, lymph node status, and high-grade tumor status (Figure S14B).Results of Kaplan-Meier curves showed that the upregulated pGli1 Ser 112 /Thr 115 /Ser 116 level exhibited negative relation to ESCC survival (Figure S14C).Specifically, analysis of 45 cases of clinical ESCC samples showed that stromal CCL22 exhibited positive relation to intratumoral expression of pGli1 Ser 112 /Thr 115 /Ser 116 (Figure S14D).

DISCUSSION
We previously reported that TAMs-released CCL22 facilitates ESCC malignancy via FAK-based signalosome. 14he present work suggested that Gli1 was activated independent of SMO via FAK/AKT pathway, where the acti-vated AKT phosphorylated Gli1 at Ser 112/ Thr 115/ Ser 116 sites, causing the dissociation of Gli1 from SuFu binding and enhancement of its activity, and resultantly inducing stemness and metastasis of ESCC.These results support that TAMs-derived CCL22 is the key chemokine for stimulating ESCC malignancy.7][38] According to our results, TAMs-released CCL22 stimulates the CCR4/FAK/AKT/Gli1 signaling in ESCC cells, providing a novel TAMs/intratumoral axis in tumor progression.Our clinical ESCC samples analyses showed that intratumoral Gli1 can serve as biomarker for evaluating ESCC malignancy and ESCC prognosis, and positively correlates to the stromal level of CCL22.
0][41][42] It remains challenging to understand crosstalk mechanisms among these signaling networks to develop targeted therapies.FAK functions as the convergence to coordinately link intracellular and intercellular signalings to facilitate tumor malignant progression. 15,17,18,43In present study, our data identified that TAMs-released CCL22 could act as a stress to stimulate Gli1 activity.Mechanistically, in response to CCL22 stimulation, FAK induced AKT to interact with Gli1 and then phosphorylate Gli1 on N-terminal Ser 112 /Thr 115 /Ser 116 sites, resulting in Gli1 release from its endogenous inhibitor-SuFu binding and enhance its transcriptional activity independent of SMO.Our observations provide the novel phosphorylation sites in Gli1 for facilitating its transcriptional activity, and extend prior studies indicating the signaling proteins-mediated Gli1 activation.Interestingly, our results showed that Gli1 did not interact with other components of CCL22/FAK signalosome, except for AKT, solidifying the result that Gli1 as a substrate for FAK/AKT pathway and the mechanisms for SMO-independent Gli1 activation.Importantly, disruption of CCL22/FAK signalosome effectively blocked the phosphorylation and activation of Gli1.Mutation of Ser 112 /Thr 115 /Ser 116 sites in Gli1 is sufficient to facilitate/abolish SuFu's inhibition against Gli1 and the signaling from upstream FAK.
FAK targeting is efficient when it is combined with additional agents for enhancing the efficacy of targeted therapy, chemotherapy, or immunotherapy in solid tumors. 18,44For identifying cases who may gain the greatest beneficial effects from FAK inhibitor-based combinatorial therapy, it is necessary to understand the interaction between FAK signaling and other druggable oncogenic proteins and relevant molecular mechanisms.HH pathway is recognized to be the therapeutic target in various tumors, such as ESCC.6][47][48] In the present work, GDC-0449 inhibit ESCC cell growth in vivo and in vitro, supporting that GDC-0449 could also be used for ESCC treatment.Furthermore, Gli1 was activated via FAK/AKT pathway independent of SMO, but it was not suppressed via SMO inhibitors and was responsible to FAK inhibitors.Safety and effectiveness of FAK inhibitors had been studied in several clinical trials; however, FAK inhibitors usually exert restricted effect on cancers as the monotherapy, but they synergistically act with cytotoxic or other targeted agents.Importantly, one Phase II study was designed for analyzing combinatorial effect of HH inhibitor-vismodegib with FAK inhibitor-GSK2256098 on progressive meningioma cases (ClinicalTrials.govIdentifier: NCT02523014, first posted:2015), providing the possibility of combination of FAK and Gli1 inhibitors in clinical tumor treatment.In our study, cotreatment FAK inhibitor with SMO inhibitor, VS-4718 with GDC-0449, exhibited the superior inhibition against cancer development, invasion, and stemness activity than each agent alone.Although SMO inhibitors display promising tumorsuppressive effect, resistance development may possibly be related to the overactivation of upstream signaling pathways.Our data of AKT phosphorylating Gli1 while enhancing its activity offers the molecular mechanism for CCL22/FAK axis-regulated and AKT-mediated Gli1 activation independent of SMO.Therefore, combined therapy of inhibitors that target both pathways is the efficient way for treating ESCC, or even other tumors.Additionally, through the IHC analysis of clinical ESCC samples, the expression of Gli1 and pGli1 Ser 112 /Thr 115 /Ser 116 was positively correlated with stromal CCL22, indicating that the above ESCC cases possibly experience canonical HH pathway as well as CCL22-regulated Gli1 activation independent of SMO.According to these clinical results, such cases cannot fully respond to GDC-0449 monotherapy; however, they can gain beneficial effects from combined therapy of inhibitors that target FAK and HH pathways.Thus, exploring a vast array of possible therapeutic combinations based on signaling crosstalk assists in the simultaneous targeting of the above pathways.
In summary, the present work identified Gli1 to be the substrate for CCL22/FAK/AKT axis, which provided the SMO-independent Gli1 activation mechanism that was stimulated by TAMs-derived chemokine.Our results also indicate that the combination of the inhibitors toward FAK and HH pathways potently suppressed ESCC with or without TAMs compared with monotherapy.Importantly, stromal CCL22 was related to intratumoral Gli1 in clinical ESCC samples, suggesting that the combined treatment that targeted FAK and HH pathways is efficient in treating ESCC, especially in the context of TME (Figure 7).

Cell culture and transfection
Human primary TAMs extracted from ESCC tissues were according to prior work. 14Human KYSE410 and KYSE510 ESCC cells (provided by Dr. Shemada from Kyoto University) were first cultured with RPMI-1640 medium that contained 1% penicillin-streptomycin as well as 10% fetal bovine serum (FBS).The Gli1 shRNA1 and shRNA2, and indicated AKT or Gli1 mutants were stably transfected into KYSE410 or KYSE510 cells, and positive cells were selected for further studies.The sequences of CCR4 siRNAs and FAK siRNA were in line with our prior works. 14,49The sequences of Gli1 shRNA1 and 2 were shown as follows: Gli1 shRNA1: 5′-GCGAGAAGCCACACAAGTGCA CGTTTGAA-3′; Gli1 shRNA2: 5′-TCTCACCTCTGTCGGATGCCAGCC TGGAC-3′.

Soft agar colony formation assay
The cell transformation assay kit (Cat# K921-100; Biovision) was utilized to analyze the anchorage-independent growth of ESCC cells.Briefly, the KYSE410/KYSE510 cell suspensions mixed by 1.2% agarose solution and 1 × DMEM solution were introduced into 96-well plate precoated with solidified agarose layer.Then, indicated agents or/and CCL22 (50 ng/mL) were treated for 8 days.Every well was introduced with DMEM solution containing WST work-ing solution, followed by 4-h incubation under 37 • C. The microplate reader (TECAN) was employed to measure OD values of all wells at 450 nm and quantitatively analyze the anchorage-independent growth ability of indicated ESCC cells.[52]

Invasion assay
ESCC cell invasive ability was evaluated by cell invasion assay kit (Cat # K912-100; Biovision).Briefly, KYSE410/KYSE510 cells were added into top transwell chamber, and the indicated agents were also added to the top well.TAMs or CCL22 (50 ng/mL) was introduced into bottom transwell chamber.After 24 h, uninvaded cells were removed out of top chamber with the cotton swab, invaded cells were obtained using cell dissociation solution, and incubated with invasion assay kit-contained cell dye at 37 • C for 60 min.The invasive cells were then quantified using a fluorometer.The exact protocol was followed as mentioned in our prior works. 14,51,52The crystal violet staining of invaded ESCC cells was also applied according to our previous studies. 14,50

Luciferase reporter assay
The Gli luciferase reporter assay is applied to evaluate the transcriptional activity of Gli.Briefly, after inoculation of KYSE410 and KYSE510 cells onto the 24-well plate, the pGLI luciferase reporter plasmid (containing multi-sites of GLI-responsive element) was transfected into indicated KYSE410 and KYSE510 cells.Subsequently, the indicated ESCC cells harbored pGLI luciferase reporter vector has been lysed by luciferase assay-related cell lysis solution and then subjected to firefly luciferase reporter assay to assess luciferase activity (measurement of the amount of conversion from luciferin to oxyluciferin).

Quantitative ELISA assays
Human CCL22/MDC ELISA kit (R&D Systems; Cat# DMD00) was applied to assess the secretion of CCL22 from TAMs (six cases).

F
I G U R E 6 Combination of VS-4718 and GDC-0449 in esophageal squamous cell carcinoma (ESCC) treatment in vivo.(A) KYSE510 (upper panel) or KYSE410 (lower panel) tumor-beared mice with (right panel)/without (left panel) rhCCL22 were treated with VS-4718 or GDC-0449 and their combination.Curves of tumor volumes were shown.(B-D) The expression of Ki-67 (B), CD31 (C), or LYVE1 (D) in indicated KYSE410 and KYSE510 tumors was evaluated using quantitative enzyme-linked immunosorbent assay (ELISA) assays.Black bar: KYSE410 tumor, left panel of KYSE410 group: cont (without C-C motif chemokine 22 [CCL22]), right panel of KYSE410 group: with CCL22; gray bar: KYSE510 tumor, left panel of KYSE510 group: cont (without CCL22), right panel of KYSE510 group: with CCL22; *p < 0.05; ***p < 0.001 as compared with the control cells.Error bars, mean ± SD of five independent experiments.

F I G U R E 7
Proposed model of C-C motif chemokine 22 (CCL22)-mediated the crosstalk between focal adhesion kinase (FAK) and glioma-associated oncogene 1 (Gli1) in tumor cells.Tumor-associated macrophages (TAMs)-released CCL22 activates intratumoral CCR4/FAK/AKT axis, which induces the phosphorylation of Gli1 Ser 112/ Thr 115/ Ser 116 sites and facilitates Gli1 releases from suppressor of fused homolog (SuFu) to enhance Gli1 activity, and then induce the stemness and metastasis of tumor cells.FAK inhibitor-VS-4718 enhanced the antitumor effect of Hedgehog (HH) inhibitor-GDC-0449 both in in vitro and in vivo.